An isoenzyme of creatine phosphokinase associated with rabbit heart mitochondria

An isoenzyme of creatine phosphokinase associated with mitochondria from rabbit heart was separated by electrophoresis on cellulose acetate membranes and detected by NADPH fluorescence. This procedure detected isoenzymes in samples containing activity as little as 0.1 IU/ml. Creatine phosphokinase activity of washed mitochondrial preparations was approximately 2.8 IU/mg protein. Mitochondria from liver or heart did not retain adsorbed activity after incubation with enzyme from sarcoplasm followed by vigorous washing. Electrophoretic migration of the isoenzyme associated with mitochondria was cathodal contrary to sarcoplasmic isoenzymes, even when mitochondrial membranes were first lysed with deoxycholate. Electrophoretic migration of sarcoplasmic isoenzymes was not altered by adsorption to intact mitochondria from liver. Molecular weight of the mitochondrial isoenzyme, estimated by Sephadex chromatography, was approximately 80,500, indicating that it did not represent monomer adsorbed to other mitochondrial constituents. Its pH optimum, sensitivity to urea, dinitrofluorobenzene, and heat inactivation differed from that of enzyme in supernatant fractions of heart homogenate. Creatine phosphokinase activity contributed to ADP regeneration from ATP as shown in polarographic studies of washed mitochondria and determinations of mitochondrial ATP hydrolysis with and without oligomycin, creatine, and Mg 2+, substantiating the presence of the phosphokinase in intact mitochondria.